Thrombin (factor IIa) is involved in a multiplicity of activating and inhibitory mechanisms of plasmatic blood coagulation and is thus the central enzyme of secondary hemostasis, which primarily comprises the formation of fibrin. Less well studied and understood is the role of thrombin in primary hemostasis, which comprises the activation of platelets and adhesion of platelets as a result of an endothelial injury. It is known that thrombin is a platelet activator and stimulates the aggregation of platelets. The most important thrombin receptors on the platelet surface are, firstly, the PAR receptors (protease-activated receptors) and, secondly, the glycoprotein Ib-V-IX receptor complex. The glycoprotein Ib-V-IX receptor complex comprises the integral membrane protein glycoprotein Ib (GPIb), the integral membrane protein glycoprotein IX (GPIX) and glycoprotein V (De Candia, E., Mechanisms of platelet activation by thrombin: A short history. Thrombosis Research 2012, 129: 250-256).
GPIb is a double-chain molecule consisting of a heavy chain with an apparent molecular mass of about 145 kDa (synonymous: alpha-chain or GPIbα) and a light chain with an apparent molecular mass of about 22 kDa (synonymous: beta-chain or GPIbβ), which are connected to one another via disulfide bonds (Lopez, J. A. et al., Cloning of the a chain of human platelet glycoprotein Ib: A transmembrane protein with homology to leucine-rich α2-glycoprotein. Proc. Natl. Acad. Sci USA 1987, 84: 5615-5619).
The GPIbα protein contains binding sites for thrombin and thus brings about the binding of thrombin to the glycoprotein Ib-V-IX receptor complex. A fragment of the GPIbα chain is glycocalicin, which is proteolytically cleaved from the intact receptor in the platelet membrane. Glycocalicin is detectable in plasma. Elevated concentrations of free glycocalicin in plasma indicate a disruption in platelet function (Beer, J. H. et al., Glycocalicin: A New Assay—The Normal Plasma Levels And Its Potential Usefulness in Selected Diseases. Blood 1994, 83(3): 691-702).
Since thrombin and platelets play a central role in the development of arterial thromboses and since inhibitors of platelet aggregation for prophylactic and therapeutic use are meanwhile being researched and used, the specific study of the interaction of thrombin and platelets is of great interest.
It is therefore desirable to have methods which allow the detection of modulators, i.e., inhibitors or activators, of platelet-thrombin interaction in patient samples. Such methods would allow the monitoring of platelet inhibitor therapies or even the detection of physiological disruptive factors, such as, for example, activating or inhibitory autoantibodies.